1. Field of the Invention
The inventions described herein are directed to the general field of anatomical pathology, and particularly to the preparation of biological samples, specifically tissue sections, for subsequent staining with chemical, immunohistochemical or in situ hybridization-based compositions. The tissue preparation methods and compositions provide for novel deparaffinization and solvent exchange of fluids within tissues, thereby readying them for further or potentially simultaneous staining.
2. Description of Related Art
The analysis of biological tissue samples is a valuable diagnostic tool used by the pathologist to diagnose many illnesses including cancer and infectious diseases and by the medical researcher to obtain information about cellular structure.
In order to obtain information from a biological tissue sample it usually is necessary to perform a number of preliminary operations to prepare the sample for analysis. While there are many variations of the procedures to prepare tissue samples for testing, these variations may be considered refinements to adapt the process for individual tissues or because a particular technique is better suited to identify a specific chemical substance or biological marker within the tissue sample. However, the basic preparation techniques are essentially the same. Biological tissue samples may derive from solid tissue such as from a tissue biopsy or may derive from liquid-based preparations of cellular suspensions such as from a smear (e.g., PAP smear), bone marrow, or cellular suspension.
Typically such procedures may include the processing of the tissue by fixation, dehydration, infiltration and embedding in paraffin wax; mounting of the tissue on a glass slide and then staining the sample; labeling of the tissue through the detection of various constituents; grid analysis of tissue sections, e.g., by an electron microscope, or the growing of sample cells in culture dishes.
Depending on the analysis or testing to be done, a sample may have to undergo a number of preliminary steps or treatments or procedures before it is ready to be analyzed for its informational content. Typically the procedures are complex and time consuming, involving several tightly sequenced steps often utilizing expensive and/or toxic materials.
For example, a typical tissue sample may undergo an optical microscopic examination so that the relationship of various cells to each other may be determined or abnormalities may be uncovered. Thus, the tissue sample must be an extremely thin strip of tissue so that light may be transmitted therethrough. The average thickness of the tissue sample or slice (often referred to as a “section”) is on the order of 2 to 10 micrometers (1 micrometer= 1/1000th of a millimeter). Typically, a tissue sample is either frozen or fixed in a material (a fixative) which not only preserves the cellular structure but also stops any further enzymatic action which could result in the putrification or autolysis of the tissue.
After fixation, the tissue sample is then dehydrated by the removal of water from the sample through the use of increasing strengths of a water-miscible alcohol, typically ethanol The alcohol then is replaced by a chemical, typically a nonpolar material, which mixes with paraffin wax or some other plastic substance impregnant which can permeate the tissue sample and give it a consistency suitable for the preparation of thin sections without disintegration or splitting. The process of removing the water, or aqueous-based solutions, and replacing it with a nonpolar material, such as a nonpolar organic solvent, is called “solvent exchange” because it involves the sequential exposure of the tissue to solvent solutions of varying proportions of water/alcohol/nonpolar organic solvent until the water in the tissue is exchanged with another fluid (or when embedding tissue, a semi-solid paraffin wax also commonly referred to as paraffin). Solvent exchange can be used in either direction, i.e., it is a 2-way process; such as the process of removing the water and replacing it with a nonpolar material, and the process of removing the nonpolar material and replacing it with water.
A microtome is then utilized to cut thin slices from the paraffin-embedded tissue sample. The slices may be on the order of 5 to 6 micrometers thick while the diameter may be on the order of 5000 to 20000 microns. The cut thin sections are floated on a water bath to spread or flatten the section. The section is then disposed on a glass slide usually measuring about 2.5 by 8 centimeters (1×3 inches).
The paraffin wax or other impregnant is then removed by solvent exchange, e.g., exposing the sample to a paraffin solvent such as xylene, toluene or limonene, the solvent then being removed by alcohol, and the alcohol removed by sequential alcohol/water mixtures of decreasing alcoholic concentrations, until eventually the tissue is once more infiltrated by water or aqueous solutions. The infiltration of the sample by water permits the staining of the cell constituents by water soluble chemical and immunochemical dyes. This process is known as a deparaffinizing process.
Certain aspects of the deparaffinizing process have been improved in recent years. Toxic paraffin solvents such as xylene and toluene are now replaceable with less toxic nonpolar organic solvents such as Terpene Oil (e.g. AMERICLEAR™, Baxter Healthcare Diagnostics, McGaw Park, Ill.), isoparaffinic hydrocarbons such as MICROCLEAR™ from Micron Diagnostics of Fairfax, Va., and HISTOLENE™, a dewaxer that is 96% d-Limonene (Fronine Pty Ltd, Riverstone, New South Wales, Australia). New automated methods have also debuted. For example, Ventana Medical Systems' U.S. Pat. No. 6,544,798 describes an automated method of removing paraffin wax from tissue sections using only hot water with surfactant. The process relies on the physical partitioning of the liquefied paraffin from the tissue by taking advantage of the immiscibility of liquefied paraffin and hot water. The process is widely used on the BENCHMARK® series of automated tissue stainers.
U.S. Pat. No. 6,632,598 (Zhang et al.) describes methods and compositions for deparaffinizing paraffin-embedded tissue. The method involves contacting a paraffin wax-embedded specimen with a dewaxing composition to solubilize the wax impregnating the specimen prior to histochemical analysis. The dewaxing compositions specifically include a paraffin-solubilizing organic solvent selected from the group consisting of aromatic hydrocarbons, terpenes and isoparaffinic hydrocarbons, a polar organic solvent, and a surfactant to solubilize the wax associated with the specimen. Compositions can further comprise water. A cited advantage of the compositions is that they do not require xylene, toluene or similar undesirable paraffin solvents. However, the actual compositions all require large amounts of polar organic solvent, typically a water-miscible alcohol.
There remains a need for improved tissue preparation processes that do not require toxic or hazardous chemicals, and methods that decrease the time and steps involved in treating tissue samples to render them acceptable for tissue staining operations.